TCP1/TCP2 Polypeptides
Transcription factors are usually defined as proteins that show sequence-specific DNA binding affinity and that are capable of activating and/or repressing transcription. The Arabidopsis thaliana genome codes for at least 1533 transcriptional regulators, accounting for ˜5.9% of its estimated total number of genes (Riechmann et al. (2000) Science 290: 2105-2109). The TCP family of transcription factors is named after its first characterized members (teosinte-branched1 (TB1), cycloidea (CYC) and PCNA factor (PCF); Cubas P et al. (1999) Plant J 18(2): 215-22). In Arabidopsis thaliana, more than 20 members of the TCP family polypeptides have been identified, and classified based on sequence similarity in the TCP domain into Class I (also called Group I or PCF group) transcription factors that positively regulate gene expression, and Class II (also called Group II or CYC-TB1 group) transcription factors that negatively regulate proliferation. All TCP transcription factors are characterized by a non-canonical predicted basic-Helix-Loop-Helix (bHLH), that is required for both DNA binding and homo- and hetero-dimerization (see Cubas et al. above).
Surprisingly, it has now been found that increasing expression in a plant of a nucleic acid sequence encoding a TCP1 or a TCP2 transcription factor gives plants having enhanced yield-related traits relative to control plants. The particular subgroup of TCP polypeptides suitable for enhancing yield-related traits is described in detail below.
According one embodiment, there is provided a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression of a nucleic acid encoding a TCP1 or a TCP2 polypeptide in a plant.
Epsin-Like Proteins
Eukaryotic cells possess an elaborate membrane system that functions in uptake of molecules (endocytosis) or in delivery of molecules to the cell exterior (secretory pathway). The secretory pathway leads from the endoplasmatic reticulum via the Golgi apparatus to the cell membrane. The endocytic pathway goes from the cell membrane to the cell interior. All these pathways make use of vesicles that budd off from the organelle where they originate from and which are highly selective with respect to the content they have and to their destination. Newly synthesised proteins need to be transported to the different subcellular locations or exported to the extracellular environment. Intracellular trafficking is controlled by many proteins, which are for example part of the vesicle, or assist in vesicle formation or fusion, or regulate the trafficking or assist in selection of cargo proteins etc. Many of these proteins are shared among plants, yeast and animals, indicating that the intracellular trafficking machinery is conserved among eukaryotes. One such group of proteins is characterised by the presence of a conserved “Epsin N-Terminal Homology” (ENTH) domain. The ENTH domain is capable of binding to phosphatidylinositols and therefore thought to play a role in targeting these proteins to specific compartments and assist in clathrin-mediated budding. ANTH (AP180 N-Terminal homology) domains are postulated to have a similar function as ENTH domains, but are part of structurally different proteins.
Epsin-like proteins all comprise an ENTH domain, and are postulated to play similar roles in clathrin-coated vesicle formation; Epsin-like proteins are reported to interact with various proteins (Lee et al., Plant Physiology 143, 1561-1575, 2007; Song et al., Plant Cell 18, 2258-2274, 2006).
Adenylate-1 PTs (AMP Isopentyltransferases/ATP/ADP Isopentyltransferases)
Phytohormones control plant growth and development, in response to endogenous and environmental stimuli. Examples of phytohormones include abscisic acid, auxins, cytokinins, ethylene, gibberellins, brassinolides, salicyclic acid, jasmonates, signalling peptides, and systemin.
In plants, naturally occurring cytokinins (CKs) constitute a group of adenine derivatives carrying either an isopentenyl side chain (isoprenoid CKs; most abundant type) or an aromatic group (aromatic CKs; rare), and play an essential role in plant development. The first and rate-limiting step of the biosynthesis of isoprenoid CKs is catalyzed by isopentenyltransferases, which transfer the isopentenyl moiety from delta(2)-dimethylallyl diphosphate (DMAPP) or hydroxymethylbutenyl diphosphate (HMBDP) to position N6 on a conjugated adenine. The isopentyltransferases can be subdivided into three subgroups, depending on which conjugated adenine they utilize:                1) AMP isopentyltransferases (also named DMAPP:AMP isopentyltransferase, EC 2.5.1.27), which preferentially use adenosine 5′-monophosphate as acceptor molecule; typical examples are found in phytopathogenic bacteria, such as in, Agrobacterium tumefaciens, Pseudomonas syringae, Pseudomonas solanacearum (Ralstonia solanacearum) and Pantoea agglomerans (Erwinia herbicola), nitrogen-fixing symbiotic cyanobacterium Nostoc, or slime mold Disctyostelium discoideum.         2) ATP/ADP isopentyltransferases (also named DMAPP:ATP/ADP isopentyltransferase), which preferentially use adenosine 5′-triphosphate or adenosine 5′-diphosphate as acceptor molecule; for example 8 ATP/ADP isopentyltransferases are found in Arabidopsis thaliana (Miyawaki et al (2006) Proc Natl Acad Sci USA 103(44): 16598-16603).        3) tRNA isopentyltransferases (also named DMAPP:tRNA isopentyltransferase, or tRNA delta(2) isopentenyl pyrophosphate transferase (IPPT), EC 2.5.1.8), which preferentially use adenine at position 37 of certain tRNAs (located in the cytoplasm, in the plastids and in the mitochondria), next to the anticodon; the enzyme has been purified and the gene cloned from bacteria, yeast, animals, and plants.        
The two first subgroups (collectively named adenylate-IPTs) catalyse the direct de novo biosynthesis of free cytokinins, essentially constituted of isopentenyladenine (iP)-types and transzeatin (tZ)-types of cytokinins. The third subgroup (named tRNA-IPTs or IPPTs) catalyses cytokinin formation by isopentenylation of tRNA, which when degraded liberates cytokinin nucleotides, which in turn will be used to biosynthesize cis-zeatin (cZ)-types of cytokinins. Thus, the rate of tRNA turnover also strongly determines the availability of free cytokinin nucleotides.
While tRNA is a common source of free cytokinins in prokaryotes (Koenig et al. (2002) J Bacteriol 184:1832-1842), both tRNA- and adenylate-IPT pathways contribute to cytokinin biosynthesis in seed plants (Miyawaki et al. (2006) Proc Natl Acad Sci USA 103(44): 16598-16603). However, the tRNA pathway is generally considered to be insufficient to account for a significant source of cytokinins in seed plants. In conclusion, the two biosynthetic pathways lead to the synthesis of different cytokinins, and in different proportions.
Both adenylate-IPTs and tRNA-IPTs have in their N-terminus the ATP/GTP P-loop binding motif (A, G)-X4-G-K-(S, T). Another well-known conserved region specific to eucaryotic tRNA-IPTs and absent in prokaryotic tRNA-IPTs, is located at the C-terminus: the Zn-finger-like motif C2H2 (C-X2-C-X(12,18)-H-X5-H. The function of Zn-finger-like motif in tRNA-IPTs is possibly in connection with protein-protein interactions and nuclear localisation (Golovko et al. (2000) Gene 258: 85-93).
When an adenylate-IPT from Agrobacterium tumefaciens was constitutively overexpressed in plants, or expressed at weaker or conditionally, these showed the typical effects of cytokinin overproduction, such as uncontrolled axillary bud growth (reduced apical dominance), the formation of small curling leaves, delayed root formation, and modified senescence (for example, Luo et al. (2005) Plant Growth Regulation 47:1-47, and references therein)
Transgenic Arabidopsis and canola plants expressing a bacterial adenylate-IPT under the control of a seed-specific promoter had an average seed yield per plant that was not significantly increased compared to control plants (Roeckel et al. (1997) Transgenic Res 6(2):133-41).
US patent application 2006/0010515 describes transgenic Arabidopsis thaliana plants expressing an adenylate-IPT from Agrobacterium tumefaciens using independently three cell-cycle regulated promoters, which plants have increased leaf size/vegetative mass, increased plant height, increased branch number, increased flower and silique number.
Short Root (SHR)
Members of the GRAS gene family (an acronym based on the designations of known genes: GAI, RGA and SCR) encode transcriptional regulators that have diverse functions in plant growth and development, such as gibberellin signal transduction, root radial patterning, axillary meristem formation, phytochrome A signal transduction, and gametogenesis. Phylogenetic analysis divides the GRAS gene family into eight subfamilies, which have distinct conserved domains and functions (Tian et al., 2004 (Plant Molecular Biology, Volume 54, Number 4, pp 519-532). GRAS proteins contain a conserved region of about 350 amino acids that can be divided in 5 motifs, found in the following order: leucine heptad repeat I, the VHIID motif, leucine heptad repeat II, the PFYRE motif and the SAW motif. SHORT ROOT, or SHR, is a member of the GRAS family of plant transcription factors and is a protein involved in root development.
Granted U.S. Pat. No. 6,927,320 B1 describes SHR genes and discloses that SHR gene expression controls cell division of certain cell types in roots, affects the organisation of root and stem, and affects gravitropism of aerial structures. It is suggested that modulation of SHR expression levels can be used to modify root and aerial structures of transgenic plants and enhance the agronomic properties of such plants. It is also suggested that plants engineered with SHR overexpression may exhibit improved vigorous growth characteristics which may be identified by examining any of the following parameters: 1. the rate of growth, 2. vegetative yield of the mature plant, 3. seed or fruit yield, 4. seed or fruit weight, 5. total nitrogen content of the plant, 6. total nitrogen content of the fruit or seed, 7. the free amino acid content of the plant, 8. the free amino acid content of the fruit or seed, 9. the total protein content of the plant, and 10. total protein content of the fruit or seed.